JPH05262661A - Production of non-covalent polysaccharide-protein association having pharmacological activity - Google Patents

Abstract

PURPOSE: To obtain a non-covalent polysaccharide-protein association having immunoregulatory and hematopoietic activities. CONSTITUTION: This association is produced by a combination of seed or seed powder and an inactive yeast or its dried extract after natural fermentation adjusted in a specific medium/extraction treatment, hydrolysis adjusted by successive chemical-enzymatic action, concentration treatment, further purification by adsorption or thorough dialysis and drying treatment.

Description

Detailed Description of the Invention

[0001]

This invention is not covalently bound (n
on-covalent) Polysaccharide-protein combination (association)
(Hereinafter referred to as Ps-Pr NCA) and their application in human and veterinary medicine in various pharmaceutical forms.

[0002]

2. Description of the Related Art Scientific and technical literature [Cancer Res., Cancer Res., 30, 2776, (197)
0): Nature such as Chihara, 222,68
7, (1989): Cancer Chemotherapy of Cancer
Chemotherapy) 1, 251 (1974): and cancer chemotherapy such as Kamei, 4, 166 (1977)] is a bacterium,
The production and pharmacological properties of polysaccharides (Ps) isolated from yeast and bacterial cultures are described. Of the other useful properties, particularly important are activities such as antitumor, antivirus, immunomodulation, hypocholesterolemia, hypolipidemia, hypoglycemia, and reticuloendothelial activator.

The direct prior art most relevant to the present invention is Spanish (ES) Patent No. 543,855, which describes a process for the production of nitrogenated polysaccharides (N-Ps). in the method for generating a fermented by two different bacterial genera Zymomonas of (Zymomonas) and two consecutive inoculation of pure cultures it was temporally separated in Pediococcus (Pediococcus). Then, decomposition-hydrolysis treatment by chemical and enzymatic action, filtration and production of N-Ps adsorbate having pharmacological activity are performed.

The known prior art methods have several drawbacks, related to the drawbacks resulting from the handling of cultures of pure microorganisms, such as the need to use sterile media, contamination, protection of the genetic integrity of the strain and treatment of the substrate. Thus, there is a drawback resulting from the limited biological capacity of a single specifically inoculated microorganism. The adverse consequences are substrate conversion variability or product biosynthesis variability, which results in lack of yield reproducibility and / or
Or, it means lack of reproducibility of the properties of the target product, which cannot be corrected by the subsequent fractionation and purification treatments.

The present invention describes a method which does not have the above-mentioned disadvantages, since the method is based on Hesseltine [CW H
esseltine Ann. Rev. Microbiol, 37 , 575-60
1 (1983)] and a "mixed culture" with the corresponding advantages and with corresponding advantages, the method being more basic than the natural fermentation method, ie pure inoculum (inocul).
a) and the expression of the method is essentially based on the pressure of biological selection produced by the special medium unique to the present invention, whereby the same natural microorganisms at the same time can be used. Fermentation standards are established by continuous and continuous growth. Furthermore, the present invention offers the advantage that the medium described above gives rise to fermentation standards and extraction characteristics so that different precursors can be used. These methods are always processed in the same way to yield the corresponding active target product. Therefore, the advantages obtained can be summarized as follows: "Mixed culture" instead of pure inoculum of laboratory strains
The method of using naturally-occurring microorganisms embodied by the selective pressure in (1) is achieved, and the risk of loss of this method due to contamination or denaturation of the genetic characteristics of the strain used is correspondingly eliminated.

2. The method, which is a mixed culture of naturally occurring strains, achieves a method capable of treating different precursors of both the polysaccharide and protein components of different target products defined as Ps-Pr NCA. This pluripotency of the method of the invention is not possible for pure inoculum-based methods, as it is possible to effectively modify only the specific substrate of the inoculum. That is, various novel pharmacologically active Ps-Pr NCA are produced.

3. Another method for purifying Ps-Pr NCA has been developed and Ps-Pr NCA can be used in another, more effective pharmaceutical dosage form.

Ps-Pr NCA have the pharmacological properties that allow them to be applied as human and veterinary medicines.

[0009]

Means for Solving the Problems, Actions and Effects The object of the present invention relates to a method for producing a polysaccharide-protein combination (Ps-Pr NCA) which is pharmacologically active and is not covalently bound, and comprises the following steps: a) A medium is produced by adding a metal oxide, a divalent metal salt, a bacteriostatic agent, elemental sulfur and a precursor that produces a protein fraction and a polysaccharide fraction to a specific volume of deionized water while stirring. Fermentation characterized by acidification of the medium, the suspension obtained after homogenization is kept at a temperature in the range 35-45 ° C. without stirring and is maintained under these conditions until a pH of 3-5 is reached. Is generated.

B) A strong inorganic or organic acid (pKa 0.1-3.0) in a pre-filtered fermentation medium by combining a chaotropic agent and a protease active in acidic medium, pH 1.0-2.8. To cause hydrolysis of controlled chemical and enzymatic actions at a temperature of 35-45 ° C. to obtain components of Ps-Pr NCA as a single macromolecule in solution: and c) removing low molecular weight contaminants. By Ps-Pr
Purify the NCA, which is i) by dialysis / exhaustive ultrafiltration with a suitable membrane to give pure Ps-Pr NCA in an authentic solution, or
ii) by precipitation in the form of an insoluble adsorbate by adding a water-miscible organic solvent in the presence of an inorganic calcium salt. Both purification methods give the products used in the manufacture of various pharmaceutical dosage forms.

The precursor material used is the desired product Ps-P.
r NCA is an empirically selected biological material that carries molecules that give rise to precursors that form part of NCA. The molecule forms part of the tissue or cell structure from which the precursor is released to be extracted and denatured. The protein fraction (Pr) -producing precursor consists of a number of naturally occurring products, including one or several seeds, grains or nuts such as rapeseed, peanuts, castor seeds, sunflower seeds, soybeans. , Rice, a degreasing residual mixture of these, or coarse powder. Polysaccharide (Ps) fraction generating precursors are inactive yeast dry, the following genera Candida (Candida) spp., Sa <br/> Kkaromisesu (Saccharomyces) genus Rhodotorula (Rhodo
Torula) genus Suporoboromisesu (Sporobolomyces) genus, c <br/> Nsenura (Hansenula) spp, selected from Pichia (Pichia) sp or their dry extracts.

For the purposes of the present invention, any possible polysaccharide fraction precursor pair (hereinafter PR-Ps) / one or more protein fraction precursors (hereinafter P
R (s) -Pr) can be used fairly from the precursors mentioned above. Each PR-Ps / PR (s) -Pr pair gives the corresponding pharmacologically active Ps-Pr NCA after fermentation treatment defined by the conditions of the medium and subsequent purification.

The object of the present invention relates to the preparation of pharmacologically active polysaccharide-protein combinations which have utility in human and veterinary medicine. To this end, a method characterized by the following technical composition is described: (a) of the protein and polysaccharide fractions of Ps-PrNCA empirically selected on the basis of the biological activity of the corresponding target product. A number of precursors from which the molecules forming the fraction are obtained.

(B) Fermentation / characterized by the property of controlling the fermentation process of naturally obtained microorganisms with organic components and extracting and denaturing the molecules forming Ps-PrNCA
Extraction medium.

(C) Ps-Pr NCA having pharmacological activity
Regulated chemical, physical and enzymatic hydrolysis conditions selected for the purpose of hydrolysing all extracted macromolecular components except those that form part of.

(D) Ps-Pr by dialysis / ultrafiltration
Formation of insoluble adsorbates that produce products used in NCA purification methods and the manufacture of various pharmaceutical dosage forms.

The process according to the invention is characterized in that it comprises the following steps: In the first step , manganese and cobalt with a specific volume of deionized water, a metal oxide such as manganese dioxide and anions from hydrochloric acid or sulfuric acid. Salt and known bacteriostats such as camphor, elemental sulfur and natural rosin (70%
A medium is produced by adding the organic acid of the abietic acid group having an acid content of 1) with stirring. After homogenization, add 3
Hold at a temperature of 5-45 ° C.

In the second step , the Ps-Pr NCA precursor is produced and added to the medium.

In the third step , the fermentation medium is heated to 35 to 45 ° C.
Hold still under controlled temperature. When a pH of 3-5 is reached, the method is held for a further 4-13 days, after which this third step of the method is considered complete and the fourth step is carried out.
In the fourth step, the liquid filtrate of the fermentation treatment has a p of 0.1 to 3.0.
Treated with a strong acid having a Ka such as hydrochloric acid, trichloroacetic acid, trifluoroacetic acid, phosphoric acid or sulfuric acid to a pH of 1 to 2.8 and a pH of 1 to 3 which can be selected from the catotropic agent and pepsin, papain, chemopapain and bromelin.
Treated with a range of active lysing enzymes, in this case 35
The hydrolysis reaction is carried out at a temperature of ~ 45 ° C for 6 to 72 hours.
Ps-Pr NCA is found in solution after the treatment.

The fifth step of the process consists of concentrating the solution from the above steps. After filtration to clean the medium, the molecular block (c
Ultrafiltration through a membrane with ut-off). In this way, the volume of the solution is reduced to a final volume of 1/3 to 1/18 of the initial volume.

In the sixth step , the product material can be purified in various ways by selective adsorption or by exhaustive dialysis and then by evaporation to dryness or drying in the presence of salts: variant ( a) : Purification by selective adsorption is carried out by adding calcium chloride, adjusting the pH of the medium and subsequently changing the polarity by adding a water-miscible organic solvent such as ethanol or acetone, which results in insoluble adsorbates. To isolate Ps-PrNCA.

Variant (b) : Purification by exhaustive dialysis is 1
This is done by diafiltering the solution from the fifth step against deionized water using a membrane with a molecular block of 50,000 to 50,000 Daltons. Evaporation to dryness by freeze-drying or spray-drying gives the desired product in soluble form.

Variant (c) : Drying in a muffle in the presence of stabilizing inorganic salts such as calcium sulphate and calcium phosphate gives an insoluble product similar to the adsorbate.

In the seventh step , the product obtained in the preceding step is processed to produce various pharmaceutical dosage forms: i) Capsules, scent bags or tablets are insoluble form of the product stabilized with an inorganic salt. (Process (a) or (c)).

Ii) Aqueous solutions, suspensions, microemulsions, suppositories, injectable solutions or with or without absorption enhancers, are prepared from the product in soluble form (variant (b)).

The products prepared as described in the methods of Examples 1 to 4 below are the basis for therapeutic use as human and veterinary pharmaceuticals and exhibit a number of pharmacological properties described below.

In serum of mice treated with bacterial endotoxin
Tumor Necrosis Factor (TNF) Concentration 25 μg / animal E. coli Endotoxin Serotype 05
5 Balb the product described in Example 1 at a dose level of 150 mg / kg for 6 consecutive days prior to intravenous administration of B5.
/ C was administered orally to Mice. The results of treatment with the product described in Example 1 show a 50% or more inhibition of serum TNF concentrations obtained 90 minutes after LPS administration. Zymosan or Calmette-Guerin
Similar results were obtained with test animals sensitized with endotoxin using E. coli. Cell line L9 for TNF measurement
This was done by measuring the cytotoxicity of serum against 29.

Hypercholesterolia treated with Triton
Cholesterol concentration reduction in hypertensive rats Intraperitoneal administration of Triton WR1239 in saline at a dose of 500 mg / kg to Wistar rats induces hypercholesterolemia. When the product described in Example 1 was added to Triton solution and administered at a dose of 500 mg / kg, a 40% decrease in serum cholesterol concentration was obtained as compared with the control group. Serum cholesterol was measured 18 hours after ip administration of the product.

Mice base corticosterone concentration
When the product described in Example 1 was orally administered to valve / c mice at a dose of 150 mg / kg, the basal cortis was measured by radioimmunoassay (radioimmu / assay) 5 hours after the administration of the product. An increase of over 200% was obtained with regard to the costerone serum concentration.

1600 mg / k of the product described in Example 2
Similar results were obtained when administered rectally at a dosage of g.

Progenitor cells (CFU-S and CFU-G
M) of haematopoietic activity evaluated by the number of
C with the product described in Example 3 at an increasing dose of 0.4 mg / kg
Intravenous administration to 57B1 / 6 rats resulted in pluripotency of the spleen (p
luripotent) cells (CFU-S) increased by more than 150% and granulocyte-macrophage line precursor splenocytes (CFU-GM) increased by more than 200%, which was administered to controls. It measured 5 days after doing.

After sublethal radiation, pluripotent cells (CFU
-S) increase in splenic hematopoietic activity assessed by increasing the number of S-) 6 consecutively after the sublethal dose of 5.5-6.0 Gy.
Oral administration of the product described in Example 4 to C57B1 / 6 rats at a dose of 150 mg / kg daily showed a 60% increase in the number of pluripotent splenocytes (CFU-S) over controls.

Immunosuppressive mau infected with monocytosis listeria
When the scan of the product of Example 4 described in a dosage of increased infection before 6 days 150 mg / kg of survival in Switzerland (Swiss) MICE administered orally,
Is silica with immunosuppressive Moreover mononucleosis Listeria (Listeria
a ) protection of rats infected with a monocytogenes) . Immunosuppression was induced by intraperitoneal treatment with 120 mg / kg silica one day before inducing infection. Protection of the rat is manifested in the treated rats as an increase in the LD ₅₀ that reached a protection rate similar to that of non-immunosuppressed rats.

Protective effect of lethal dose of radiation on maize C57B1 / in a single intravenous dose of 0.4 mg / kg 1 day before receiving lethal dose of radiation (7.75 Gy)
When administered at 6 mice, the product prepared in Example 3 gave 100% viability as assessed 15 days later.

Analytical data for various products prepared by operating up to step 6, including the methods described in Examples 1-4, are shown in Table I.

[0036] (1) Dubois (Dubois A. et al. Anal. Chem. 28)
350, (1956)).

(2) Lowry (Lowry OH, J. Biol.
Chem. 193 , 265, (1951)).

The polysaccharide fraction has a molecular weight of 10 ^{4 to} 3 × 10 ⁵ , 1-6 and / or 1-2, 1-3 or 1
Mannose with a -4 bond (70-90% w / w),
Glucose (5-20% w / w) and galactose (0
~ 3% w / w) produced by the heteropolysaccharide produced. The protein fraction has a molecular weight of 10 ^{4 to} 2 × 10 ⁵ .

The infrared (IR) spectra (KBr) of the non-covalently bonded Ps-Pr combinations prepared according to Examples 1 to 4 are given in Figures 1 to 4, respectively. These infrared spectra are 1000 cm ⁻¹ , variable slot resolution, spectral noise of 0.5% T, and 4000-6 in 6 minutes.
Obtained using potassium bromide tablets at a sample concentration of 2% in a Perkin-Elmer Model 881 infrared spectrophotometer, scanning in the range of 000 cm ^-1 . Infrared spectra are shown electronically corrected by the Savitzky / Golay method and the absorbance is automatically magnified.

[0040]

Example 1 First step: Production of medium 0.2-0.4 g of manganese dioxide, 5.2-7.1 g of manganese sulphate, 2-5 g of rosin and 2 precipitated in a 2 l reactor. 0.4-4.2 g of cobalt chloride, 4-6 g of camphor and 4.5-6.5 g of sulfur were added to 1,000 ml of deionized water. The mixture was kept at a temperature of 35-45 ° C.

Second Step: Preparation and Addition of Precursor 5 to 25 g of soybean which had been washed and soaked in water in advance was crushed and added to the medium prepared in the first step. Then 3
~ 12g of dead, dried Candida utilis (C
andida u tilis) was added.

Third step: Fermentation-extraction The fermentation mixture is mixed until the pH of the medium reaches 3.0 to 5.0.
Hold at rest at a temperature of 5 to 45 ° C.
Hold for days.

Fourth step: hydrolysis by chemical and enzymatic action The reaction medium is filtered through a layer of diatomaceous earth in a Buchner funnel, 85% phosphoric acid is added to the filtrate and 1.0-2. 8
PH was reached. Then 6-12 g of urea and 0.
1-0.6 g of pepsin was added. After homogenization, the mixture was held static at 35-45 ° C for 6-72 hours.

Fifth Step: Concentration The reaction mixture from the fourth step was filtered through a layer of diatomaceous earth in a Buchner funnel and 10,000-50,000.
Concentrated by ultrafiltration through a polysulfone membrane with Dalton molecular block. The volume of the reaction mixture was thus reduced to about 1/5 of the original volume.

Sixth step: Variant (a): Adsorbate 10-25 g of anhydrous calcium chloride are added to the concentrate of the fifth step, after which p is added by addition of a 10% solution of NaOH.
H was adjusted to 3.5-4.4. This solution was poured onto acetone, and the final proportion of acetone was 25-40% (volume / volume).
Volume). The resulting mixture was allowed to stand for 24 hours, after which most of the supernatant was decanted. The precipitate is filtered on a Buchner funnel, 25-40% (v / v)
Was thoroughly washed with acetone / water mixture and dried in oven with ventilation.

Seventh step: Pharmaceutical dosage form The product produced in the sixth step is treated with CaHPO ₄ .2H until the total sugar concentration measured by the Dubois method is about 1.2 to 2.0% (w / w). ₂ O / CaSO ₄・ 2H ₂ O 2
Diluted with a mixture of 1/1.

Example 2 First Step: Production of Medium The medium was the same as that described in Example 1.

Second Step: Preparation and Addition of Precursor Similarly, 5-25 soybeans washed, soaked in water and ground.
g was used. In this case, the polysaccharide source is 3-12 g
Inert dry brewer's yeast (Saccharomyces cerevisi
al ).

Third step: Fermentation-extraction The fermentation mixture was worked up in the same way as in Example 1.

Step 4: Hydrolysis by chemical and enzymatic action Filtration, acidification and hydrolysis were carried out in the same way as described in Example 1.

Fifth step: Concentration The filtered reaction medium was concentrated by ultrafiltration under the same conditions as described in Example 1. Final capacity is 1 of the initial capacity
It was / 5.

Sixth Step: Variant (b): Soluble Product The concentrate from the fifth step was subjected to exhaustive dialysis by ultrafiltration and a concentration cycle by addition of deionized water to the original volume. Dialysis was allowed to proceed until the permeate had a conductivity similar to that of deionized water. The resulting solution was then concentrated by ultrafiltration to a volume of 1/6 to 1/10 of the original volume and finally freeze dried.

Step 7: Pharmaceutical formulation Supocire BP, a suppository containing 0.125% (w / w) in a labrafil type excipient, was prepared from this lyophilized product.

Example 3 First Step: Production of Medium The method was the same as described in Examples 1 and 2.

Second Step: Preparation and Addition of Precursor Soybeans 2.9-1 washed and soaked in water and then ground
A mixture of 4.6 g of castor seeds 2.1-10.4 g with an inert dry Candida u tilis was used.

Third step: Fermentation-extraction The fermentation mixture was worked up as in Example 1.

Step 4: Hydrolysis by chemical and enzymatic action Same as described in Examples 1 and 2.

Fifth Step: Concentration The procedure was as described in Example 1 and the solution was concentrated 6 times.

Sixth Step, Variant (b): Soluble Product Same as described in Example 2.

Seventh Step: Pharmaceutical Formulation The product from the sixth step was immediately dissolved in a serum solution of salt for intravenous injection.

Example 4 Steps 1 to 5 In the same manner as in Example 3, the solution was concentrated to 1/6 of the original volume.

Sixth step, modified method (a): adsorbate The same as in Example 1.

Seventh Step: Pharmaceutical Dosage Form As described in Example 1.

[Brief description of drawings]

1 is a graph showing an infrared spectrum curve of a non-covalently bonded Ps-Pr combination of the present invention prepared according to Example 1. FIG.

2 is a Ps- of the present invention produced according to Example 2. FIG.
It is a graph showing the infrared spectrum curve of Pr NCA.

FIG. 3 shows the Ps- of the present invention produced according to Example 3.
It is a graph showing the infrared spectrum curve of Pr NCA.

4 is a Ps- of the present invention produced according to Example 4. FIG.
It is a graph showing the infrared spectrum curve of Pr NCA.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location A61K 35/78 T 7180-4C U 7180-4C C12P 19/04 B 7432-4B 21/06 8214- 4B // (A61K 31/715 37:02) (A61K 35/72 35:78) 7180-4C (72) Inventor Rafael de la Citi Boville Spain. 28042-Madrid. Kanoa. 8 (72) Inventor Jose Antonio Matsuchi Tsudeyuri, Spain. 28043 Madrid. Sera Magna. 8 (72) Inventor Giuan Pablo Liber Ranieri Spain. 28014-Madrid. Gutenberg. 8

Claims (12)

[Claims] 1. The next step, namely, (a) one or several precursors of a protein fraction (Pr) selected from oily seeds, grains or nuts, and a polysaccharide fraction (from a yeast or its extract ( One or several precursors of Ps) are reacted with each other at a temperature of 35 to 45 ° C., in which case cobalt and manganese compounds, elemental sulfur, abietic acid or abietic acid salts and a bacteriostatic agent are selected. The above reaction shall be carried out using an aqueous medium for extraction-fermentation adjusted to a constant temperature, and the resulting mixture shall be held stationary in the absence of bacterial inoculum: (b) after which pKa0.1 A Ps-Pr solution obtained by mixing an acid having 2.0, a chaotropic agent, and an enzyme selected from the group consisting of pepsin, papain, chemopapain or bromelin with 1.0 to .0
Subject to selective and controlled chemical and enzymatic hydrolysis at a pH of 35 to 45 ° C .: and (c) adding a protic or aprotic water-miscible organic solvent to the reaction medium. Or by dialysis-ultrafiltration to precipitate the Ps-Pr adsorbate in the presence of salt from the filtered concentrate to isolate pure Ps-Pr combinations and to produce various pharmaceutical formulations. , A method for producing a novel polysaccharide-protein (Ps-Pr) combination which is not covalently bound. 2. The following composition: metal oxide, in particular 0.2-0.
Manganese dioxide at a concentration of 4 g / l: 1.9-each
Manganese and cobalt salts with anions from hydrochloric acid or sulfuric acid at concentrations of 2.6 g / l and 4.0-6.0 g / l:
4.5-6.5 g / l concentration elemental sulfur and 2.0-
The process according to claim 1, wherein an extraction-fermentation aqueous medium is prepared having an organic acid from the group consisting of abietic acid in the natural form of rosin having an acid content of 70% at a concentration of 5.0 g / l. 3. A seed, a grain from the group consisting of rapeseed seed, peanut, castor bean, sunflower seed, soybean, rice, defatted residue or coarse powder thereof as a naturally occurring precursor product which produces a protein fraction. Alternatively, selecting nuts and selecting an inert dry yeast from Candida, Saccharomyces, Rhodotorula, Sporoboromyces, Hansenula, Pichia or a dried raw extract thereof as a precursor for producing a polysaccharide fraction. The method according to item 1. 4. The amount of precursor substance added to the extraction-fermentation medium is from 5.0 to 25 g / l for seeds and for the dry weight equivalent of the coarse powder, and for the precursor substance of the polysaccharide fraction 3. It is 0 to 12.0 g / l.
The method described. 5. The extraction-fermentation medium is kept static at a temperature of 35-45 ° C. without stirring, and the fermentation process has a pH of 3.
A process according to claim 1 wherein a solution containing a molecule which forms a non-covalently bound polysaccharide-protein combination is obtained by holding it for 4 to 13 days while adjusting it to 0 to 5.0. 6. Hydrolysis by chemical and enzymatic action comprises the following components in the filtered medium: hydrochloric acid, phosphoric acid, sulfuric acid, trichloroacetic acid or trifluoromethanesulfonic acid to bring the pH to 1 to 2.8. Acid selected from the group: 6.0
A chaotropic agent consisting of urea at a concentration of ~ 12.0 g / l: by adding a degrading enzyme selected from the group consisting of papain, chemopapain, pepsin or bromelin in an amount of 0.1-0.6 g / l: Hydrolysis reaction is 3
The method according to claim 1, which proceeds at a temperature of 5 to 45 ° C for 6 to 72 hours. 7. The concentration of the reaction solution is 10,000 to 50,0.
A method according to claim 1 performed by a membrane having a molecular block in the range of 00 Daltons. 8. A polysaccharide-protein combination which is not covalently bound is purified in an insoluble adsorbate form, the acid used for the decomposition at that time is phosphoric acid, and calcium chloride is added to 10.
Add to final concentration of 0-25.0 g / l, add sodium hydroxide solution to pH of 3.5-4.4, add water-miscible solvent such as ethanol or acetone to 25-4.
The method of claim 1 wherein said purification is carried out with addition to a final concentration of 0% w / w. 9. The non-covalently linked polysaccharide-protein combination is purified by exhaustive dialysis through a membrane with a molecular block of 10,000 to 50,000, followed by evaporation to dryness, lyophilization, spray drying or A process according to claim 1 isolated by concentration and drying in the presence of salts such as calcium sulfate and calcium phosphate. 10. A non-covalently linked polysaccharide-protein combination having immunomodulatory activity and pharmacological activity for hematopoietic recovery is administered in a pharmaceutical dosage form selected for its use as a human and veterinary drug. The method described. 11. A non-covalently linked polysaccharide-protein combination produced in response to a combination of one or more selected protein fraction precursors with a selected polysaccharide fraction to provide the desired pharmacologically active combination. The method according to claim 1, wherein 12. The following groups: Candida-soybean Candida-castor candida-soybean: castor Saccharomyces-soybean Saccharomyces-castor Saccharomyces-castor: soybean Ps-Pr combinations, their adsorbates and salts. The method of claim 1 wherein a mixture of